Interferon proteins have been classified into three types, alpha, beta and gamma (abbreviated to IFN-.alpha., IFN-.beta. and IFN-.gamma. respectively) based on antigenic and structural differences. Gamma interferon has a number of characteristics that differentiate it from alpha and beta interferons. Among these differences are antigenic distinctiveness and greater activity with regard to immunoregulation and anti-tumor effects. Human gamma interferon (referred to herein as "h-IFN-.gamma.") may be produced by T lymphocytes stimulated by mutagens or by antigens to which they are sensitized. It may also be obtained through cloning and expression techniques now well known to the art.
Recently, it has become possible by the progress in genetic engineering to produce many physiologically active polypeptides from microorganisms or animal cells, although these substances have been produced by separation and purification from an organism. However, it cannot yet be said that a method has been established for extracting and purifying the intended substance with a purity sufficient to be used for drugs and without causing denaturation or decomposition.
Gamma interferon-containing cells, however obtained, are collected and are disrupted by various means such as osmotic shock, ultrasonic vibration, grinding or high shear disruption and the disrupted cell-gamma interferon mixture is then processed to isolate the gamma interferon. The insoluble debris is separated by centrifugation and the gamma interferon-containing supernatant is collected for purification.
Although disclosure has been made of certain technology for such production methods, e.g. a method extracting and purifying the polypeptide produced by recombinant microorganism by using guanidine hydrochloride and urea (Japanese Patent Public Disclosure No. 161321/1984 and U.S. Pat. No. 4,476,049) and a purification method using a monoclonal antibody (Japanese Patent Public Disclosure No. 186995/1984), the intended substance is not always adequately purified without being subjected to denaturation and without its activity being lost.
European Patent Application 0,087,686 discloses a three-step process for purifying human immune interferions from the cell-free supernatant or extract from the crude interferon source. In the first step (for naturally occurring interferon), an affinity column, such as Concanavalin-A Sepharose is used, followed by chromatography on a carboxymethyl silica column using an increasing salt gradient and finally, on a silica gel permeation column. If sufficient purity is not obtained, concentration and chromatography on either the TSK or CM column is used.
European Patent Application 0,063,482 disclosed a purification process employing chromatographic methods using (1) Controlled Pore Glass beads; (2) Concanavalin-A Sepharose; (3) Heparin-Sepharose or Procion Red-agarose; and (4) gel filtration.
European Patent Applications 0,107,498 and 0,077,670 disclose a purification scheme employing (1) polyethyleneimine precipitation; (2) pH precipitation of bacterial proteins; (3) concentration and dialysis; (4) chromatography on (a) carboxymethyl cellulose; (b) a calcium phosphate gel; (c) a carboxymethyl cellulose; and (d) gel filtration resins.
These purification processes require a multitude of steps, cause degradation of the interferon by degradation or aggregation of the interferon molecule, or otherwise result in a gamma interferon product obtained in low yield or with low activity.
It goes without saying that a method for extracting and purifying the intended polypeptide from the culture mixture of the intended substance-producing microorganism without the activity of the intended substance being lost and without being accompanied by denaturation is important for such used as pharmaceuticals and that the establishment of such technology is useful from the viewpoint of industry.
Such a purification method has been particularly desired for interferon, the employment of which in pharmaceuticals is now proceeding. Interferons have anti-virus activity, but IFN-.gamma. is expected to be useful as an anti-tumor agent and immune regulator because of its particularly strong cell growth inhibition. Furthermore, interferon activity has several specificities; for example, when interferon is used as a pharmaceutical, it is preferable to use interferon which originated from a human. Furthermore, it is desirable to establish processes for extracting and purifying interferon produced by genetic engineering.
Usually, in extracting and purifying a polypeptide obtained from recombinant microorganisms, cultured microorganisms are first killed by using a bactericide (a necessary process from the viewpoint of safety) and then the dead cells are disrupted and subsequently subjected to extraction. In these treatments, the intended polypeptide is sometimes denaturated and its activity may be lost. Furthermore, these treatments are apt to activate proteases included in the cells and sometimes decompose the intended polypeptide.
A method in which a protein denatured and solubilized with a denaturing agent such as urea or guanidine hydrochloride is extracted and the denaturing agent is removed in the course of purification has previously been disclosed for polypeptides purified from cells or recombinant microorganisms (as described before in Japanese Patent Public Disclosure No. 161321/1984, U.S. Pat. No. 4,476,049, etc.). However, it is difficult to safely obtain complete renaturation of the intended polypeptide even though the denaturing agents are removed. Therefore, this method is not preferred if the intended polypeptide is used as a pharmaceutical because, when the partially denatured polypeptide is mixed, it can become an antigen. On the other hand, in the purification method using a monoclonal antibody which has also been reported (as described before in Japanese Patent Public Disclosure No. 186995/1984, etc.), it can be thought that a denatured and undesirable polypeptide or a polymerized polypeptide such as dimer and trimer may be bonded to the monoclonal antibody, depending upon the antigenic determinant recognized with the monoclonal antibody used. Recently, a method for extracting h-IFN-.gamma. produced by recombinant Escherichia coli in the presence of a protease inhibitor for the purpose of inhibiting decomposition of polypeptide with protease has been disclosed in the above mentioned U.S. Patent, but the guanidine hydrochloride used therein is also known as a protein denaturing agent (see, example, Japanese Patent Public Disclosure No. 161321/1984). It is therefore expected that, although the decomposition of polypeptide with protease can be inhibited, production of a denatured protein may well result.
It would be further desirable to (1) provide a purification scheme to separate gamma interferon from the cell debris of the disrupted cells in which the gamma interferon was produced; (2) separate gamma interferon from cell contaminants in high yields and with high purity and activity; (3) separate recombinant gamma interferon from cell contaminants; and (4) separate gamma interferon from cell contaminants without substantially degrading the interferon. The purification process described below is such a process.